Rapid development of ultrasound contrast agents in the recent years has generated a number of different formulations which are useful in ultrasound imaging of organs and tissue of human or animal body. These agents are designed to be used primarily as intravenous or intra-arterial injectables in conjunction with the use of medical echographic equipment. These instruments typically group B-mode image formation (based on the spatial distribution of backscatter tissue properties) and Doppler signal processing (based on Continuous Wave or pulsed Doppler processing of ultrasonic echoes to determine blood or liquid flow parameters). Other ultrasound imaging methods could also benefit from these agents in the future, such as Ultrasound Computed Tomography (measuring attenuation in transmission), or Diffraction Computed Tomography (measuring scattering and attenuation parameters in angular reflection). Based on suspensions of gas microbubbles in aqueous liquid carriers, these injectable formulations may basically be divided into two categories: Aqueous suspensions in which the gas microbubbles are bounded by the gas/liquid interface, or an evanescent envelope involving the molecules of the liquid and a surfactant loosely bound at the gas to liquid interface and suspensions in which the inicrobubbles have a material boundary or a tangible envelope formed of natural or synthetic polymers. In the latter case the microbubbles are referred to as microballoons. There is yet another kind of ultrasound contrast agents: suspensions of porous particles of polymers or other solids which carry gas microbubbles entrapped within the pores of the microparticles. These contrast agents are considered here as a variant of the microballoon kind. Although physically different, both kinds of gas microbubbles when in suspension are useful as ultrasonic contrast agents. More on these different formulations may be found in EP-A-0 077 752 (Schering), EP-A-0 123 235 (Schering), EP-A-0 324 938 (Widder et al.), EP-A-0 474 833 (Schneider et al.), EP-A-0 458 745 (Bichon et al.), U.S. Pat. No. 4,900,540 (Ryan), U.S. Pat. No. 5,230,882 (Unger), etc.
Certain of the above mentioned ultrasound contrast agents are developed and commercially available, while others are at different stages of clinical trials. However, whether commercially available or on clinical tests, these products all suffer from problems linked with storage. The problems of storage are intrinsic to suspensions which, due to their very nature, undergo phase separation or segregation, gas bubble aggregation, gas diffusion and, after long periods, even precipitation of various additives. Segregation of the gas microbubbles or microballoons comes from the fact that the suspensions are typically made of uncalibrated microbubbles whose sizes vary from about 1 .mu.m up to about 50 .mu.m. A vast majority of the gas bubbles in the known suspensions is found to be between 1 .mu.m and about 10 .mu.m. Due to the microbubble size distribution, these suspensions, during storage, undergo segregation in which larger microbubbles migrate to the top while the smaller ones concentrate in the lower parts, often leading to complete phase separation. Attempts to resolve this problem through the use of viscosity enhancing agents have shown that the rate of segregation may be reduced but not eliminated.
Gas microbubble aggregation is a process during which the larger bubbles absorb the smaller ones, thus growing in size. With phase separation, this process accelerates and suspensions with microbubbles having average size of e.g. between 2 .mu.m and 8 .mu.m after a while may evolve in suspensions with microbubble size between e.g. 5 .mu.m to 12 .mu.m or larger. This is particularly undesirable in cases where suspensions of calibrated microbubbles and suspensions intended for opacification of the left heart are concerned. The change in size does not only alter the echogenic properties of the contrast agent, but also renders the agent inapplicable for certain applications such as those based on the passage of the microbubbles through the lungs. Microbubbles with sizes over 10 .mu.m are unlikely to pass through the lung capillaries and therefore, in addition to creating hazardous conditions such suspensions are less suitable for imaging of the left heart.
Another problem with gas suspensions and their storage comes from the gas diffusion which occurs at relatively low rates, but which accelerates with phase separation. Inevitable escape of the gas from the microbubble suspensions is thus further aggravated and in extreme cases may lead to complete gas depletion of the medium. Hence, the combined effect of these various mechanisms on destruction of the gas suspensions therefore results in a very rapid degradation of the agent.
In some ultrasound imaging approaches, one of the desirable aspects of these contrast agents is for the microbubbles or gas-containing particles to be distributed within a tight size window. The reason is given hereafter. The effectiveness of these agents to increase the contrast in images produced by medical ultrasonographic equipment is based, primarily, on greatly enhanced scattering of the incoming ultrasonic energy, and secondly, on modified attenuation properties of the tissues containing these agents. By contrast, it is meant a measure of the relative signal amplitude obtained from regions to be perfused by the contrast agent compared with the signal amplitude from regions not receiving the contrast agent. By enhancement, it is meant an increase in the contrast value observed following administration of the contrast agent, compared to the contrast observed prior administration. As mentioned earlier, the type of imaging equipment most directly benefitting from these agents is the family of echographic instruments (B-mode or Doppler). The different attenuation properties of tissues containing the agent compared to those not containing the agent can also be exploited to improve the diagnostic value of the imaging procedure. Furthermore, the ultrasonic-frequency dependence of both the scattering and attenuation properties of the agent can be exploited to increase spatial tissue discrimination further. In these cases, the physical laws governing this frequency dependence systematically depend on the microbubble- or particle-size. Thus, the algorithms used are more efficient when acting on echoes originating from microbubbles or particles with tight size-distribution. As an example, one such approach exploits non-linear oscillation of microbubbles to detect echo frequency-components at the second-harmonic of the fundamental excitation frequency. Since the tissues not containing the contrast agent do not exhibit the same non-linear behaviour as the microbubbles, this method is able to enhance significantly the contrast between the regions containing and those not containing the contrast agent. This enhancement is more pronounced, for a given particle-count per unit volume, when the sizes are narrowly distributed. However, the preparation of products with such narrow-size distributions is time consuming; providing a ready supply of these calibrated suspensions would facilitate greatly the further development and use of this technique. Reliable storage of such preparations with unchanged size distributions is thus also of great interest.
Further difficulties with storage of aqueous gas suspensions is experienced with ultrasound contrast agents which contain phospholipids as stabilisers of the gas microbubbles. Due to hydrolysis of phospholipids, the concentration of the stabilizer (surfactant), during storage, is constantly diminished, causing loss of the microbubble content and degradation of echogenic properties of the suspension. Thus so far the problem of storage of ultrasound contrast agents comprising gas microbubble suspensions remains open.
Cold storage of aqueous gas suspensions by freezing has been known for quite some time in the food industry. For example, U.S. Pat. No. 4,347,707 (General Foods Corp.) discloses storage of gasified ice with high gas content and good storage stability by a method in which the gasified ice is prepared by contacting an aqueous liquid with hydrate-forming gases under pressure and at a temperature such that the gas hydrate complex suspended in the liquid is formed and the temperature and pressure are controllably lowered to produce e.g. carbonated ice with 85-110 ml of CO.sub.2 /g. According to the document, the gasified ice has high gas content, prolonged storage stability, is suitable for commercial distribution in the frozen state and, when placed in water, provides vigorous effervescence.
It follows that freezing of the gas suspensions in order to store them for prolonged periods of time, and reuse the preserved suspensions when necessary would not be suitable for ultrasonic contrast agents because the suspended gas has a tendency to escape from the carrier medium during defrosting. Further difficulty with the frozen gas suspensions of microbubbles lies in the fact that the expansion of the carrier medium during the freezing creates internal forces which simply destroy or crush the microbubble envelope liberating the entrapped gas and letting it escape either during storage or later during defrosting of the suspension. This problem is particularly severe for the suspensions of microbubbles having material or tangible envelopes.